Process for preparation of anhydrous azithromycin

ABSTRACT

The present invention provides a stable form of azithromycin derivatives that act as antibiotics. These compounds are in anhydrous form and have increased stability over the hydrated forms.

PRIORITY OF INVENTION

This application is a continuation under 37 C.F.R. 1.53(b) of U.S.application Ser. No. 10/373,349 filed Feb. 24, 2003, which is acontinuation under 35 U.S.C. 111 (a) of International Application No.PCT/IB01/01523 filed Aug. 23, 2001 and published as WO 02/15842 A2 onFeb. 28, 2002, which claims priority from U.S. Provisional ApplicationNo. 60/227,341, filed 23 Aug. 2000, which applications and publicationare incorporated herein by reference.

BACKGROUND OF THE INVENTION

Azithromycin is a well-known semi-synthetic macrolide antibiotic. It isprepared through the ring expansion to incorporate a nitrogen atom inthe macrolide ring of erythromycin A, followed by reductive methylation.This provides an antibiotic having more stability and greatereffectiveness than erythromycin-A.

The ring expansion and subsequent conversion of erythromycin-A toprovide azithromycin is described in U.S. Pat. No. 4,474,768, (e.g.,Example 3). Generally, the synthesis requires several steps. The productobtained is one of the hydrated versions, either monohydrate ordihydrate.

Azithromycin monohydrate is hygroscopic and thus, difficult to maintainin the monohydrated form. U.S. Pat. No. 4,963,531 and EP application 298650 teach a process for preparing azithromycin dihydrate. The processrequires preparing a solution of azithromycin monohydrate intetrahydrofuran and water. The azithromycin dihydrate is obtained bycrystallization upon addition of hexane.

In U.S. Pat. No. 4,963,531 it is disclosed that on storage at lowhumidity the azithromycin dihydrate loses water. In addition, samples ofazithromycin mono- and di-hydrate stored at higher humidity rapidlyabsorbed water. Thus, the water percentage (percent hydration) in thecrystals can vary depending on the relative humidity during storage.This variability of the percent hydration can make it difficult toaccurately determine the proper amount of active ingredient needed toprepare various dosage forms.

Thus, there is a need for forms of azithromycin that exhibit stabilityand less variability in the level of hydration.

SUMMARY OF THE INVENTION

The present invention provides a stable form of azithromycin andanalogues thereof. These compounds are in anhydrous form that exhibitsincreased stability over the corresponding hydrated form. Accordingly,there is provided an anhydrous compound of Formula I:

wherein R₁ represents hydrogen, (C₁-C₆)-alkyl, (C₆-C₁₀)-aryl or(C₇-C₁₆)-aralkyl wherein the R₂, R₃, R₄, R₅ and R₆ groups individuallyare hydrogen or (C₁-C₆)alkyl. The present invention also provides aprocess for preparing a compound of Formula I. The anhydrous compound ofFormula I is prepared by a process comprising removal of an organicsolvent from a solution comprising a hydrated form of the compound ofFormula I in the organic solvent or a solution of the hydrated compoundof Formula I in a mixture of the organic solvent and water so as toprovide the anhydrous compound.

The solvents that are useful in practicing the present invention includeany solvent that is capable of co-distilling with water or forming anazeotrope with water. Non-limiting examples of suitable solvents includealcohols, haloalkanes, esters, ethers or aromatic solvents. Examples ofsuitable solvents for practicing the invention are C₃-C₆ alcohols suchas, for example, n-propanol, 2-propanol, n-butanol, 2-butanol,n-pentanol, 2-pentanol, 3-pentanol and the like; or halo(C₁-C₆)alkanessuch as, for example, methylene chloride, chloroform, carbontetrachloride, 1,1,1-trichloroethylene, 1,1,2-trichlorethylene and thelike; esters such as, for example, methyl acetate, ethyl acetate and thelike; ethers such as, for example, tetrahydrofuran, tetrahydropyran andthe like.

The invention also provides a pharmaceutical composition comprising ananhydrous compound of Formula I, or a pharmaceutically acceptable saltthereof, in combination with a pharmaceutically acceptable diluent orcarrier.

Additionally, the invention provides a method for treating a microbialinfection in a mammal, such as a human, which comprises administering,to a mammal an antimicrobially effective amount of a compound of FormulaI in a suitable dosage form.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the Infrared spectrum of the anhydrous azithromycinof the invention.

FIG. 2 illustrates the Infrared spectrum of azithromycin dihydrate.

FIG. 3 illustrates the DSC spectrum of the anhydrous azithromycin of theinvention.

FIG. 4 illustrates the DSC spectrum of azithromycin dihydrate.

FIG. 5 illustrates the XRD spectrum of the anhydrous azithromycin of theinvention.

FIG. 6 illustrates the XRD spectrum of azithromycin dihydrate.

FIG. 7 illustrates the process for preparing compounds of Formula I.

DETAILED DESCRIPTION

The following definitions are used, unless otherwise described: halo isfluoro, chloro, bromo, or iodo. Alkyl denotes both straight and branchedgroups; but reference to an individual radical such as “propyl” embracesonly the straight chain radical, a branched chain isomer such as“isopropyl” being specifically referred to. Aryl denotes a phenylradical or an ortho-fused bicyclic carbocyclic radical having about nineto ten ring atoms in which at least one ring is aromatic.

It will be appreciated by those skilled in the art that compounds of theinvention having a chiral center may exist in and be isolated inoptically active and racemic forms. Some compounds may exhibitpolymorphism. It is to be understood that the present inventionencompasses any racemic, optically-active, polymorphic, orstereoisomeric form, or mixtures thereof, of a compound of theinvention, which possess the useful properties described herein, itbeing well known in the art how to prepare optically active forms (forexample, by resolution of the racemic form by recrystallizationtechniques, by synthesis from optically-active starting materials, bychiral synthesis, or by chromatographic separation using a chiralstationary phase) and how to determine nicotine agonist activity usingthe standard tests described herein, or using other similar tests whichare well known in the art.

Specific and preferred values listed below for radicals, substituents,and ranges, are for illustration only; they do not exclude other definedvalues or other values within defined ranges for the radicals andsubstituents

Specifically, (C₁-C₆)alkyl can be methyl, ethyl, propyl, isopropyl,butyl, iso-butyl, sec-butyl, n-pentyl, 2-pentyl, 3-pentyl, or hexyl;halo(C₁-C₆)alkyl can be iodomethyl, bromomethyl, chloromethyl,fluoromethyl, trifluoromethyl, 2-chloro-ethyl, 2-fluoroethyl,2,2,2-trifluoroethyl, or pentafluoroethyl; C₃-C₆ alcohols can be1-hydroxypropane, 2-hydroxypropane, 3-hydroxypropane, 1-hydroxybutane,2-hydroxybutane, 1-hydroxypentane, 2-hydroxypentane, 1-hydroxyhexyl, or6-hydroxyhexane and the like; aryl can be phenyl, indenyl, or naphthyl.

A specific value for R₁ is CH₃.

A specific value for each of R₂, R₃, R₄, R₅ and R₆ is hydrogen.

A preferred group of compounds are compounds of formula I; or apharmaceutically acceptable salt thereof.

Another preferred group of compounds are compounds of formula I whereinR₁ is a lower alkyl group having from 1 to 4 carbon atoms and each ofR₂, R₃, R₄, R₅ and R₆ is hydrogen.

A preferred compound of the invention is a compound of where R₁ ismethyl and each of R₂, R₃, R₄, R₅ and R₆ is hydrogen or apharmaceutically acceptable salt thereof.

The solvents that are useful in practicing the present invention includesolvents that remove water from a solution either by co-distillation orazeotropic distillation. These solvents will remove small amounts ofwater that are difficult to remove using standard recrystallizationtechniques.

The preferred alcohol solvents for practicing the present invention aren-propanol, 2-propanol, n-butanol or 2-butanol. Most preferred is2-propanol.

The preferred haloalkane solvents for practicing the present inventionare methylene chloride, chloroform, and carbon tetrachloride. Mostpreferred is chloroform.

The preferred ester solvents for practicing the present invention areesters such as, for example, methyl acetate, ethyl acetate and the like.The preferred ester is ethyl acetate.

The preferred ether solvents for practicing the present invention areethers such as, for example, tetrahydrofuran, tetrahydropyran and thelike. The preferred ether is tetrahydrofuran.

The preferred aromatic solvents for practicing the present invention arearomatic compounds such as benzene, toluene, xylene and the like. Thepreferred aromatic solvent is toluene.

The hydrated compounds useful in practicing the invention can beprepared according to the procedures disclosed in U.S. Pat. Nos.4,328,334, 4,474,768 and 4,517,359. The process for preparing compoundsof Formula I is illustrated in FIG. 7. Compound I, wherein R₂-R₆ are asdefined above is converted to the corresponding oxime, 2 using anexcess, e.g., about 10 equivalents, of hydroxyl amine. The oxime isrearranged via the Beckmann rearrangement with methane sulfonyl chlorideat low temperature to furnish amide, 3. The amide, 3 is then reduced,with hydrogen and a catalyst or with a metal hydride, such as sodiumborohydride to furnish amine, 4. The amine is then alkylated, e.g.,using formaldehyde in the presence of formic acid to form the methylanalogue or by alkylation methods known in the art to form otheranalogues. The product is crystallized from alcohol/water to provide thehydrated compound of Formula I.

A preferred compound of the present invention, Azithromycin, isrepresented below:

The anhydrous compounds of Formula I can be formulated as pharmaceuticalcompositions and administered to a mammalian host, such as a humanpatient in a variety of forms adapted to the chosen route ofadministration, i.e., orally or parenterally, by intravenous,intramuscular, topical or subcutaneous routes.

Thus, the present compounds may be systemically administered, e.g.,orally, in combination with a pharmaceutically acceptable vehicle suchas an inert diluent or an assimilable edible carrier. They may beenclosed in hard or soft shell gelatin capsules, may be compressed intotablets, or may be incorporated directly with the food of the patient'sdiet. For oral therapeutic administration, the active compound may becombined with one or more excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. Such compositions and preparations shouldcontain at least 0.1% of active compound. The percentage of thecompositions and preparations may, of course, be varied and mayconveniently be between about 2 to about 60% of the weight of a givenunit dosage form. The amount of active compound in such therapeuticallyuseful compositions is such that an effective dosage level will beobtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the active compound maybe incorporated into sustained-release preparations and devices.

The active compound may also be administered intravenously orintraperitoneally by infusion or injection. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, triacetin, andmixtures thereof and in oils. Under ordinary conditions of storage anduse, these preparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form should be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, the preferred methods of preparationare vacuum drying and the freeze drying techniques, which yield a powderof the active ingredient plus any additional desired ingredient presentin the previously sterile-filtered solutions.

For topical administration, the present compounds may be applied in pureform, i.e., when they are liquids. However, it will generally bedesirable to administer them to the skin as compositions orformulations, in combination with a dermatologically acceptable carrier,which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the present compounds can be dissolved or dispersed ateffective levels, optionally with the aid of non-toxic surfactants.Adjuvants such as fragrances and additional antimicrobial agents can beadded to optimize the properties for a given use. The resultant liquidcompositions can be applied from absorbent pads, used to impregnatebandages and other dressings, or sprayed onto the affected area usingpump-type or aerosol sprayers.

The amount of the compound, or an active salt or derivative thereof;required for use in treatment will vary not only with the particularsalt selected but also with the route of administration, the nature ofthe condition being treated and the age and condition of the patient andwill be ultimately at the discretion of the attendant physician orclinician.

In general, however, a suitable oral or parenteral dose will be in therange of from about 1 to about 200 mg per kilogram body weight of therecipient per day, preferably in the range of 5 to 100 mg/kg/day, mostpreferably in the range of 5 to 50 mg/kg/day.

The compound is conveniently administered in unit dosage form; forexample, containing 25 to 3000 mg, conveniently 100 to 2000 mg, mostconveniently, 250 to 600 mg of active ingredient per unit dosage form.

The invention will now be illustrated by the following non-limitingExamples.

EXAMPLE 1 Erythromycin-A Oxime

A solution of 1.40 Kg of hydroxylamine hydrochloride in isopropylalcohol and water was prepared. Sodium hydroxide, 0.81 Kg, was added inportions, at temperature of about 20° C. After the addition, the pH wasadjusted to 7.0 with acetic acid. Erythromycin base, 1.5 Kg, was added,and the solution maintained at 45-55° C. for 28 hours.

The reaction mixture was cooled to room temperature and the reactionterminated by the addition of ammonia-water mixture. The crude productwas treated with water to remove inorganic salts and furnish the titleproduct as a white crystalline material, 1.40 Kg.

EXAMPLE 2 9a-Aza-9a-homoerythromycin-A

The title product, prepared in Example 1, 1.25 Kg, was dissolved inacetone and water and maintained at a temperature of 0-5° C. The pH ofthe reaction mixture was adjusted to about 2.5 to about 2.8 withhydrochloric acid. Sodium bicarbonate, 0.48 Kg, was added in portions tothe cooled reaction mixture. After addition of the sodium bicarbonate,methane sulfonyl chloride, 0.5 Kg, was added. The reaction mixture wasstirred for 1 hour at a temperature of 0-5° C. the pH of the reactionmixture was adjusted with aqueous sodium hydroxide and the title productwas filtered off as a white crystalline material in high purity. Yield1.00 Kg.

EXAMPLE 3 9-Deoxo-9a-aza-9a-homoerythromycin-A

The title product, prepared in Example 2, 1.00 Kg, was stirred inmethanol and water. Sodium borohydride, 1 Kg, was added over four hours.The temperature was maintained below 5° C. After completion of thesodium borohydride addition, the reaction mixture was stirred for anadditional six hours at <5° C. and for an additional twenty-four hours,at room temperature. The reaction was terminated by the addition ofwater and chloroform. The chloroform layer was separated and fresh waterwas added. The product was extracted with chloroform by pH adjustmentusing dilute hydrochloric acid and sodium hydroxide.

Initially, the mixture (water and chloroform) was stirred at pH 2.5 to2.8 for 1 hour. The pH of the water layer was adjusted to 9.5 to 9.8,and the mixture was stirred for one-half hour. This sequence wasrepeated with additional chloroform three times. The water layer wasseparated and an additional portion of chloroform was added and theextraction repeated one additional time. After the extraction, thechloroform extracts were combined, dried over potassium carbonate,filtered and used in the next step without additional treatment.

EXAMPLE 4 9-Deoxo-9a-methyl-9a-aza-9a-homoerythromycin-A

The title product, prepared in Example 3, was treated with formaldehyde,0.17 L, and formic acid, 0.105 L, the reaction mixture was stirred forfour hours, under nitrogen and heated at reflux for twelve hours. Thereaction was cooled, treated with water and the pH was adjusted to 4.0to 4.5. Chloroform was added and the mixture stirred and the chloroformlayer separated. The aqueous layer was adjusted to pH 6.0 to 6.5 andextracted twice with chloroform. Additional chloroform was added to theaqueous layer and the pH was adjusted with stirring to about pH 2.0 to3.0 with dilute hydrochloric acid. The mixture was stirred vigorouslyand the chloroform layer was separated. The pH was adjusted to about pH6.0-6.5 with dilute sodium hydroxide and extracted twice withchloroform.

This sequence above was repeated five times on the aqueous layer. Thechloroform layers were combined, dried over K₂CO₃ and concentrated undervacuum. The solid residue was dissolved in isopropyl alcohol and thetitle product crystallized by adding water. The yield of azithromycinwas 0.55 Kg.

EXAMPLE 5 Azithromycin Dihydrate

The title product, 0.5 Kg, prepared in Example 4, was dissolved inwater, making the solution acidic (pH of 2.5 to 5.0) with dilutehydrochloric acid. After 20 minutes stirring the pH was raised withdilute sodium hydroxide and the solution was stirred for twelve hours.The product was crystallized as a white crystalline material in highpurity. Yield: 0.48 Kg.

EXAMPLE 6 Anhydrous Azithromycin

The azithromycin dihydrate, prepared in Example 5, or the azithromycinmonohydrate, prepared in Example 4, about 500 g, was dissolved inisopropanol, 3 L. The solution was heated and the alcohol was distilledto remove the water. After the solvent was removed the residue was driedunder vacuum to provide the anhydrous azithromycin. Yield 470 g;Purity≧96%.

EXAMPLE 7 Anhydrous Azithromycin

The azithromycin dihydrate, prepared in Example 5, or the azithromycinmonohydrate, prepared in Example 4, ˜100 g, was dissolved in chloroformand water, 1.7 L (0.7:1). The solution was heated and the solvent wasdistilled off. After the solvent was removed the residue was dried undervacuum to provide the anhydrous azithromycin. Yield 94 g; Purity≧96%.

All publications, patents, and patent documents cited in thespecification are incorporated by reference herein, as thoughindividually incorporated by reference. In the case of anyinconsistencies, the present disclosure, including any definitionstherein will prevail. The invention has been described with reference tovarious specific and preferred embodiments and techniques. However, itshould be understood that many variations and modifications may be madewhile remaining within the spirit and scope of the invention.

1-20. (canceled)
 21. A stable, pure anhydrous azithromycin having purity 96% w/w or more and having no detectable azithromycin hydrated forms when stored at 40±2° C. and at 75±5% relative humidity for a period of seven days.
 22. A stable, pure anhydrous azithromycin having purity 96% w/w or more and having no detectable azithromycin hydrated forms when stored at 25±2° C. and at 60±5% relative humidity for a period of seven days.
 23. The anhydrous azithromycin of claim 21 or 22, having X-Ray Powdered Diffraction pattern as shown in FIG.
 5. 24. The anhydrous azithromycin of claim 21 or 22, having Fourier Transform Infrared spectrum as shown in FIG.
 1. 25. The anhydrous azithromycin of claim 21 or 22, having Differential Scanning Calorimetric Thermogram as shown in FIG.
 3. 26. A pharmaceutical composition comprising the stable anhydrous azithromycin of claim 21 or
 22. 27. The pharmaceutical composition of claim 26, wherein the composition is administered by oral, parenteral, intravenous, intramuscular, topical or subcutaneous routes.
 28. A method to treast microbial infection in a mammal comprising administering an antimicrobially effective amount of the stable, pure anhydrous azithromycin of claim 21 or 22 to a mammal in need thereof. 